Abstract:

The present invention relates to novel hydrazide-containing compounds of
Formula I or Formula II, including pharmaceutically acceptable salts,
esters, or prodrugs thereof which inhibit serine protease activity,
particularly the activity of hepatitis C virus (HCV) NS3-NS4A protease.
Consequently, the compounds of the present invention interfere with the
life cycle of the hepatitis C virus and are also useful as antiviral
agents. The present invention further relates to pharmaceutical
compositions comprising the aforementioned compounds for administration
to a subject suffering from HCV infection. The invention also relates to
methods of treating an HCV infection in a subject by administering a
pharmaceutical composition comprising the compounds of the present
invention.

7. A pharmaceutical composition comprising therapeutically effective
amount of a compound according to claims 1, a pharmaceutically acceptable
salt, ester, or prodrug thereof alone, or in combination with a
pharmaceutically acceptable carrier or excipient.

8. A method of treating a hepatitis C viral infection in a subject,
comprising administering to the subject a pharmaceutical composition
according to claim 6.

9. A method of inhibiting the replication of hepatitis C virus, the method
comprising contacting the hepatitis C virus with an inhibitory amount of
a compound of claim 1.

14. A pharmaceutical composition of claim 13 wherein said additional
anti-hepatitis C virus agent is selected from the group consisting of:
α-interferon, β-interferon, ribavarin, and adamantine.

15. Compound of claim 1 wherein said compound is in a substantially pure
form.

Description:

TECHNICAL FIELD

[0001]The present invention relates to novel hepatitis C virus (HCV)
protease inhibitor compounds having antiviral activity against HCV, which
are also useful in the treatment of HCV infections. More specifically,
the invention relates to novel, hydrazide-containing HCV protease
inhibitor compounds, compositions containing such compounds and methods
for using the same, as well as processes for making such compounds.

BACKGROUND OF THE INVENTION

[0002]HCV is the principal cause of non-A, non-B hepatitis and is an
increasingly severe public health problem both in the developed and
developing world. It is estimated that the virus infects over 200 million
people worldwide, surpassing the number of individuals infected with the
human immunodeficiency virus (HIV) by nearly five-fold. HCV infected
patients, due to the high percentage of individuals inflicted with
chronic infections, are at an elevated risk of developing cirrhosis of
the liver, subsequent hepatocellular carcinoma and terminal liver
disease. HCV is the most prevalent cause of hepatocellular cancer and
cause of patients requiring liver transplantations in the western world.

[0003]There are considerable barriers to the development of anti-HCV
therapeutics, which include, but are not limited to, the persistence of
the virus, the genetic diversity of the virus during replication in the
host, the high incident rate of the virus developing drug-resistant
mutants, and the lack of reproducible infectious culture systems and
small-animal models for HCV replication and pathogenesis. In a majority
of cases, given the mild course of the infection and the complex biology
of the liver, careful consideration must be given to antiviral drugs,
which are likely to have significant side effects.

[0004]Only two approved therapies for HCV infection are currently
available. The original treatment regimen generally involves a 3-12 month
course of intravenous interferon-α (IFN-α), while a new
approved second-generation treatment involves co-treatment with
IFN-α and general antiviral nucleoside mimics like ribavirin. Both
of these treatments suffer from interferon related side effects as well
as low efficacy against HCV infections. There exists a need for the
development of effective antiviral agents for treatment of HCV infection
due to the poor tolerability and disappointing efficacy of existing
therapies.

[0005]In a patient population where the majority of individuals are
chronically infected and asymptomatic, and the prognoses are unknown, an
effective drug would desirably possess significantly fewer side effects
than the currently available treatments. The hepatitis C non-structural
protein-3 (NS3) is a proteolytic enzyme required for processing of the
viral polyprotein and consequently viral replication. Despite the huge
number of viral variants associated with HCV infection, the active site
of the NS3 protease remains highly conserved, thus making its inhibition
an attractive mode of intervention. Recent success in the treatment of
HIV with protease inhibitors supports the concept that the inhibition of
NS3 is a key target in the battle against HCV.

[0006]HCV is a flaviridae type RNA virus. The HCV genome is enveloped and
contains a single strand RNA molecule composed of circa 9600 base pairs.
It encodes a polypeptide comprised of approximately 3010 amino acids.

[0007]The HCV polyprotein is processed by viral and host peptidase into 10
discreet peptides, which serve a variety of functions. There are three
structural proteins: C, E1 and E2. The P7 protein is of unknown function
and is comprised of a highly variable sequence. There are six
non-structural proteins. NS2 is a zinc-dependent metalloproteinase that
functions in conjunction with a portion of the NS3 protein. NS3
incorporates two catalytic functions (separate from its association with
NS2): a serine protease at the N-terminal end, which requires NS4A as a
cofactor, and an ATP-ase-dependent helicase function at the carboxyl
terminus. NS4A is a tightly associated but non-covalent cofactor of the
serine protease.

[0008]The NS3-NS4A protease is responsible for cleaving four sites on the
viral polyprotein. The NS3-NS4A cleavage is autocatalytic, occurring in
cis. The remaining three hydrolyses, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B
all occur in trans. NS3 is a serine protease, which is structurally
classified as a chymotrypsin-like protease. While the NS3 serine protease
possesses proteolytic activity by itself, the HCV protease enzyme is not
an efficient enzyme in terms of catalyzing polyprotein cleavage. It has
been shown that a central hydrophobic region of the NS4A protein is
required for this enhancement. The complex formation of the NS3 protein
with NS4A seems necessary to the processing events, enhancing the
proteolytic efficacy at all of the sites.

[0010]The present invention relates to novel hydrazide-containing
compounds including pharmaceutically acceptable salts, esters, or
prodrugs thereof which inhibit serine protease activity, particularly the
activity of hepatitis C virus (HCV) NS3-NS4A protease. Consequently, the
compounds of the present invention interfere with the life cycle of the
hepatitis C virus and are also useful as antiviral agents. The present
invention further relates to pharmaceutical compositions comprising the
aforementioned compounds for administration to a subject suffering from
HCV infection. The invention also relates to methods of treating an HCV
infection in a subject by administering a pharmaceutical composition
comprising the compounds of the present invention.

[0011]In one embodiment of the present invention there are disclosed
compounds represented by Formula I or Formula II, or pharmaceutically
acceptable salts, esters, or prodrugs thereof:

[0177]A first embodiment of the invention is a compound represented by
either Formula I or Formula II, as described above, or a pharmaceutically
acceptable salt, ester or prodrug thereof, alone or in combination with a
pharmaceutically acceptable carrier or excipient.

[0178]Representative subgenera of the invention include, but are not
limited to compounds of Formula III or Formula IV:

[0211]Representative subgenera of the invention also include, but are not
limited to compounds of Formula V or Formula VI:

[0212]Wherein A, L, j, k, V, R81, R32, R83, and R84
are all as previously defined;Representative subgenera of the invention
also include, but are not limited to compounds of Formula VII or Formula
VIII:

[0216]A further embodiment of the present invention includes
pharmaceutical compositions comprising any single compound delineated
herein, or a pharmaceutically acceptable salt, ester, or prodrug thereof,
with a pharmaceutically acceptable carrier or excipient.

[0217]Yet another embodiment of the present invention is a pharmaceutical
composition comprising a combination of two or more compounds delineated
herein, or a pharmaceutically acceptable salt, ester, or prodrug thereof,
with a pharmaceutically acceptable carrier or excipient.

[0219]According to an additional embodiment, the pharmaceutical
compositions of the present invention may further contain other HCV
protease inhibitors.

[0220]According to yet another embodiment, the pharmaceutical compositions
of the present invention may further comprise inhibitor(s) of other
targets in the HCV life cycle, including, but not limited to, helicase,
polymerase, metalloprotease, and internal ribosome entry site (IRES).

[0221]According to another embodiment, the present invention includes
methods of treating hepatitis C infections in a subject in need of such
treatment by administering to said subject a therapeutically effective
amount of the pharmaceutical compounds or compositions of the present
invention. The methods can further include administration of an
additional therapeutic agent, including another antiviral agent or an
anti-HCV agent. The additional agent can be co-administered, concurrently
administered or sequentially administered with the compound or
composition delineated herein. The methods herein can further include the
step of identifying that the subject is in need of treatment for
hepatitis C infection. The identification can be by subjective (e.g.,
health care provider determination) or objective (e.g., diagnostic test)
means.

[0222]An additional embodiment of the present invention includes methods
of treating biological samples by contacting the biological samples with
the compounds of the present invention.

[0223]Yet a further aspect of the present invention is a process of making
any of the compounds delineated herein employing any of the synthetic
means delineated herein.

Definitions

[0224]Listed below are definitions of various terms used to describe this
invention. These definitions apply to the terms as they are used
throughout this specification and claims, unless otherwise limited in
specific instances, either individually or as part of a larger group.

[0226]The terms "C2-C6 alkenyl," or "C2-C8 alkenyl,"
as used herein, denote a monovalent group derived from a hydrocarbon
moiety containing from two to six, or two to eight carbon atoms having at
least one carbon-carbon double bond by the removal of a single hydrogen
atom. Alkenyl groups include, but are not limited to, for example,
ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and
the like.

[0227]The term "C2-C6 alkynyl," or "C2-C8 alkynyl," as
used herein, denote a monovalent group derived from a hydrocarbon moiety
containing from two to six, or two to eight carbon atoms having at least
one carbon-carbon triple bond by the removal of a single hydrogen atom.
Representative alkynyl groups include, but are not limited to, for
example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

[0228]The term "C3-C8-cycloalkyl", or
"C3-C12-cycloalkyl," as used herein, denotes a monovalent group
derived from a monocyclic or polycyclic saturated carbocyclic ring
compound by the removal of a single hydrogen atom, respectively. Examples
of C3-C8-cycloalkyl include, but not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and
examples of C3-C12-cycloalkyl include, but not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl,
and bicyclo[2.2.2]octyl.

[0229]The term "C3-C8-cycloalkenyl", or
"C3-C12-cycloalkenyl" as used herein, denote a monovalent group
derived from a monocyclic or polycyclic carbocyclic ring compound having
at least one carbon-carbon double bond by the removal of a single
hydrogen atom. Examples of C3-C8-cycloalkenyl include, but not
limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl,
cycloheptenyl, cyclooctenyl, and the like; and examples of
C3-C12-cycloalkenyl include, but not limited to, cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,
and the like.

[0230]The term "aryl," as used herein, refers to a mono- or polycyclic
carbocyclic ring system having one or more aromatic rings including, but
not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and
the like.

[0231]The term "arylalkyl," as used herein, refers to a C1-C3
alkyl or C1-C6 alkyl residue attached to an aryl ring. Examples
include, but are not limited to, benzyl, phenethyl and the like.

[0232]The term "heteroaryl," as used herein, refers to a mono- or
polycyclic (e.g. bi-, or tri-cyclic or more), fused or non-fused,
aromatic radical or ring having from five to ten ring atoms of which one
or more ring atom is selected from, for example, S, O and N; zero, one or
two ring atoms are additional heteroatoms independently selected from,
for example, S, O and N; and the remaining ring atoms are carbon, wherein
any N or S contained within the ring may be optionally oxidized.
Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl,
pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,
isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,
isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

[0233]The term "heteroarylalkyl," as used herein, refers to a
C1-C3 alkyl or C1-C6 alkyl residue attached to a
heteroaryl ring. Examples include, but are not limited to,
pyridinylmethyl, pyrimidinylethyl and the like.

[0234]The term "heterocycloalkyl," as used herein, refers to a
non-aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic
group fused system, where (i) each ring contains between one and three
heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii)
each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has
0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may
optionally be oxidized, (iv) the nitrogen heteroatom may optionally be
quaternized, and (iv) any of the above rings may be fused to a benzene
ring. Representative heterocycloalkyl groups include, but are not limited
to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl,
isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and
tetrahydrofuryl.

[0236]In accordance with the invention, any of the aryls, substituted
aryls, heteroaryls and substituted heteroaryls described herein, can be
any aromatic group. Aromatic groups can be substituted or unsubstituted.

[0237]It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl and
cycloalkenyl moiety described herein can also be an aliphatic group, an
alicyclic group or a heterocyclic group. An "aliphatic group" is
non-aromatic moiety that may contain any combination of carbon atoms,
hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and
optionally contain one or more units of unsaturation, e.g., double and/or
triple bonds. An aliphatic group may be straight chained, branched or
cyclic and preferably contains between about 1 and about 24 carbon atoms,
more typically between about 1 and about 12 carbon atoms. In addition to
aliphatic hydrocarbon groups, aliphatic groups include, for example,
polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and
polyimines, for example. Such aliphatic groups may be further
substituted. It is understood that aliphatic groups may be used in place
of the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene
groups described herein.

[0238]The term "alicyclic," as used herein, denotes a monovalent group
derived from a monocyclic or polycyclic saturated carbocyclic ring
compound by the removal of a single hydrogen atom. Examples include, but
not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Such alicyclic groups may
be further substituted.

[0239]It will be apparent that in various embodiments of the invention,
the substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, arylalkyl, heteroarylalkyl, and
heterocycloalkyl are intended to be divalent or trivalent. Thus,
alkylene, alkenylene, and alkynylene, cycloaklylene, cycloalkenylene,
cycloalkynylene, arylalkylene, hetoerarylalkylene and heterocycloalkylene
groups are to be included in the above definitions, and are applicable to
provide the formulas herein with proper valency.

[0240]The terms "halo" and "halogen," as used herein, refer to an atom
selected from fluorine, chlorine, bromine and iodine.

[0241]The compounds described herein contain one or more asymmetric
centers and thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)-, or as D- or L- for amino acids. The
present invention is meant to include all such possible isomers, as well
as their racemic and optically pure forms. Optical isomers may be
prepared from their respective optically active precursors by the
procedures described above, or by resolving the racemic mixtures. The
resolution can be carried out in the presence of a resolving agent, by
chromatography or by repeated crystallization or by some combination of
these techniques, which are known to those skilled in the art. Further
details regarding resolutions can be found in Jacques, et al.,
Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). When
the compounds described herein contain olefinic double bonds or other
centers of geometric asymmetry, and unless specified otherwise, it is
intended that the compounds include both E and Z geometric isomers.
Likewise, all tautomeric forms are also intended to be included. The
configuration of any carbon-carbon double bond appearing herein is
selected for convenience only and is not intended to designate a
particular configuration unless the text so states; thus a carbon-carbon
double bond depicted arbitrarily herein as trans may be cis, trans, or a
mixture of the two in any proportion.

[0242]The term "subject" as used herein refers to a mammal. A subject
therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea
pigs, and the like. Preferably the subject is a human. When the subject
is a human, the subject may be referred to herein as a patient.

[0243]As used herein, the term "pharmaceutically acceptable salt" refers
to those salts of the compounds formed by the process of the present
invention which are, within the scope of sound medical judgment, suitable
for use in contact with the tissues of humans and lower animals without
undue toxicity, irritation, allergic response and the like, and are
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well known in the art. For example, S. M. Berge, et
al. describes pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in
situ during the final isolation and purification of the compounds of the
invention, or separately by reacting the free base function with a
suitable organic acid. Examples of pharmaceutically acceptable include,
but are not limited to, nontoxic acid addition salts are salts of an
amino group formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or
with organic acids such as acetic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other methods used
in the art such as ion exchange. Other pharmaceutically acceptable salts
include, but are not limited to, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,
lauryl sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,
pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, stearate, succinate, sulfate, tartrate,
thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the
like. Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate, nontoxic
ammonium, quaternary ammonium, and amine cations formed using counterions
such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate,
alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

[0244]As used herein, the term "pharmaceutically acceptable ester" refers
to esters of the compounds formed by the process of the present invention
which hydrolyze in vivo and include those that break down readily in the
human body to leave the parent compound or a salt thereof. Suitable ester
groups include, for example, those derived from pharmaceutically
acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,
cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl
moiety advantageously has not more than 6 carbon atoms. Examples of
particular esters include, but are not limited to, formates, acetates,
propionates, butyrates, acrylates and ethylsuccinates.

[0245]The term "pharmaceutically acceptable prodrugs" as used herein
refers to those prodrugs of the compounds formed by the process of the
present invention which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower animals
with undue toxicity, irritation, allergic response, and the like,
commensurate with a reasonable benefit/risk ratio, and effective for
their intended use, as well as the zwitterionic forms, where possible, of
the compounds of the present invention. "Prodrug", as used herein means a
compound, which is convertible in vivo by metabolic means (e.g. by
hydrolysis) to afford any compound delineated by the formulae of the
instant invention. Various forms of prodrugs are known in the art, for
example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier
(1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic
Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of
Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191
(1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38
(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988);
Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems,
American Chemical Society (1975); and Bernard Testa & Joachim Mayer,
"Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And
Enzymology," John Wiley and Sons, Ltd. (2002).

[0246]Combinations of substituents and variables envisioned by this
invention are only those that result in the formation of stable
compounds. The term "stable", as used herein, refers to compounds which
possess stability sufficient to allow manufacture and which maintains the
integrity of the compound for a sufficient period of time to be useful
for the purposes detailed herein (e.g., therapeutic or prophylactic
administration to a subject).

[0247]The synthesized compounds can be separated from a reaction mixture
and further purified by a method such as column chromatography,
high-performance liquid chromatography, or recrystallization. As used
herein, the term "substantially pure" for a compound refers to the
physical state of said compound after being obtained from a purification
process or processes described herein or that are well known to the
skilled artisan, in sufficient purity to be characterizable by standard
analytical techniques described herein or as are well known to the
skilled artisan.

[0248]In one embodiment, a substantially pure compound comprises a
compound of greater than about 75% purity. This means that the compound
does not contain more than about 25% of any other compound. In one
embodiment, a substantially pure compound comprises a compound of greater
than about 80% purity. This means that the compound does not contain more
than about 20% of any other compound. In one embodiment, a substantially
pure compound comprises a compound of greater than about 85% purity. This
means that the compound does not contain more than about 15% of any other
compound. In one embodiment, a substantially pure compound comprises a
compound of greater than about 90% purity. This means that the compound
does not contain more than about 10% of any other compound. In another
embodiment, a substantially pure compound comprises a compound of greater
than about 95% purity. This means that the compound does not contain more
than about 5% of any other compound. In another embodiment, a
substantially pure compound comprises greater than about 98% purity. This
means that the compound does not contain more than about 2% of any other
compound. In one embodiment, a substantially pure compound comprises a
compound of greater than about 99% purity. This means that the compound
does not contain more than about 1% of any other compound.

[0249]As can be appreciated by the skilled artisan, further methods of
synthesizing the compounds of the formulae herein will be evident to
those of ordinary skill in the art. Additionally, the various synthetic
steps may be performed in an alternate sequence or order to give the
desired compounds. In addition, the solvents, temperatures, reaction
durations, etc. delineated herein are for purposes of illustration only
and one of ordinary skill in the art will recognize that variation of the
reaction conditions can produce the desired bridged macrocyclic products
of the present invention. Synthetic chemistry transformations and
protecting group methodologies (protection and deprotection) useful in
synthesizing the compounds described herein are known in the art and
include, for example, those such as described in R. Larock, Comprehensive
Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and
Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for
Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons
(1995).

[0250]The compounds of this invention may be modified by appending various
functionalities via any synthetic means delineated herein to enhance
selective biological properties. Such modifications are known in the art
and include those which increase biological penetration into a given
biological system (e.g., blood, lymphatic system, central nervous
system), increase oral availability, increase solubility to allow
administration by injection, alter metabolism and alter rate of
excretion.

Pharmaceutical Compositions

[0251]The pharmaceutical compositions of the present invention comprise a
therapeutically effective amount of a compound of the present invention
formulated together with one or more pharmaceutically acceptable
carriers. As used herein, the term "pharmaceutically acceptable carrier"
means a non-toxic, inert solid, semi-solid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. Some
examples of materials which can serve as pharmaceutically acceptable
carriers are sugars such as lactose, glucose and sucrose; starches such
as corn starch and potato starch; cellulose and its derivatives such as
sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil; safflower
oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a
propylene glycol; esters such as ethyl oleate and ethyl laurate; agar;
buffering agents such as magnesium hydroxide and aluminum hydroxide;
alginic acid; pyrogen-free water; isotonic saline; Ringer's solution;
ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the composition, according to the
judgment of the formulator. The pharmaceutical compositions of this
invention can be administered to humans and other animals orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, or drops),
buccally, or as an oral or nasal spray.

[0253]Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions may be formulated according to the known art using
suitable dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution,
suspension or emulsion in a nontoxic parenterally acceptable diluent or
solvent, for example, as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids such
as oleic acid are used in the preparation of injectables.

[0254]The injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which can be
dissolved or dispersed in sterile water or other sterile injectable
medium prior to use.

[0255]In order to prolong the effect of a drug, it is often desirable to
slow the absorption of the drug from subcutaneous or intramuscular
injection. This may be accomplished by the use of a liquid suspension of
crystalline or amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution, which,
in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered drug
form is accomplished by dissolving or suspending the drug in an oil
vehicle. Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of drug to polymer
and the nature of the particular polymer employed, the rate of drug
release can be controlled. Examples of other biodegradable polymers
include poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping the drug in liposomes or
microemulsions which are compatible with body tissues.

[0256]Compositions for rectal or vaginal administration are preferably
suppositories which can be prepared by mixing the compounds of this
invention with suitable non-irritating excipients or carriers such as
cocoa butter, polyethylene glycol or a suppository wax which are solid at
ambient temperature but liquid at body temperature and therefore melt in
the rectum or vaginal cavity and release the active compound.

[0257]Solid compositions of a similar type may also be employed as fillers
in soft and hard-filled gelatin capsules using such excipients as lactose
or milk sugar as well as high molecular weight polyethylene glycols and
the like.

[0258]The active compounds can also be in micro-encapsulated form with one
or more excipients as noted above. The solid dosage forms of tablets,
dragees, capsules, pills, and granules can be prepared with coatings and
shells such as enteric coatings, release controlling coatings and other
coatings well known in the pharmaceutical formulating art. In such solid
dosage forms the active compound may be admixed with at least one inert
diluent such as sucrose, lactose or starch. Such dosage forms may also
comprise, as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such a
magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise buffering
agents. They may optionally contain opacifying agents and can also be of
a composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in
a delayed manner. Examples of embedding compositions, which can be used,
include polymeric substances and waxes.

[0259]Dosage forms for topical or transdermal administration of a compound
of this invention include ointments, pastes, creams, lotions, gels,
powders, solutions, sprays, inhalants or patches. The active component is
admixed under sterile conditions with a pharmaceutically acceptable
carrier and any needed preservatives or buffers as may be required.
Ophthalmic formulation, ear drops, eye ointments, powders and solutions
are also contemplated as being within the scope of this invention.

[0261]Powders and sprays can contain, in addition to the compounds of this
invention, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these
substances. Sprays can additionally contain customary propellants such as
chlorofluorohydrocarbons.

[0262]Transdermal patches have the added advantage of providing controlled
delivery of a compound to the body. Such dosage forms can be made by
dissolving or dispensing the compound in the proper medium. Absorption
enhancers can also be used to increase the flux of the compound across
the skin. The rate can be controlled by either providing a rate
controlling membrane or by dispersing the compound in a polymer matrix or
gel.

[0263]According to the methods of treatment of the present invention,
viral infections are treated or prevented in a subject, such as a human,
by administering to the subject a therapeutically effective amount of a
compound of the invention, in such amounts and for such time as is
necessary to achieve the desired result. The term "therapeutically
effective amount" of a compound of the invention, as used herein, means a
sufficient amount of the compound so as to decrease the viral load in a
subject and/or decrease the subject's HCV symptoms. As is well understood
in the medical arts a therapeutically effective amount of a compound of
this invention will be at a reasonable benefit/risk ratio applicable to
any medical treatment.

[0264]It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided by
the attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular patient
will depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the specific
compound employed; the specific composition employed; the age, body
weight, general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of the
specific compound employed; the duration of the treatment; drugs used in
combination or contemporaneously with the specific compound employed; and
like factors well known in the medical arts.

[0265]The total daily dose of the compounds of this invention administered
to a human or other animal in single or in divided doses can be in
amounts, for example, from 0.01 to 50 mg/kg body weight or more usually
from 0.1 to 25 mg/kg body weight. Single dose compositions may contain
such amounts or submultiples thereof to make up the daily dose. In
general, treatment regimens according to the present invention comprise
administration to a patient in need of such treatment from about 10 mg to
about 1000 mg of the compound(s) of this invention per day in single or
multiple doses.

[0267]Lower or higher doses than those recited above may be required.
Specific dosage and treatment regimens for any particular patient will
depend upon a variety of factors, including the activity of the specific
compound employed, the age, body weight, general health status, sex,
diet, time of administration, rate of excretion, drug combination, the
severity and course of the disease, condition or symptoms, the patient's
disposition to the disease, condition or symptoms, and the judgment of
the treating physician.

[0268]Unless otherwise defined, all technical and scientific terms used
herein are accorded the meaning commonly known to one with ordinary skill
in the art. All publications, patents, published patent applications, and
other references mentioned herein are hereby incorporated by reference in
their entirety.

Abbreviations

[0269]Abbreviations used in the descriptions of the schemes and the
examples that follow are: [0270]aq. for aqueous; [0271]CDI for
1,1'-carbonyldiimidizole; [0272]DBU for
1,8-diazabicyclo[5.4.0]undec-7-ene; [0273]DCM for dichloromethane;
[0274]DIAD for diisopropyl azodicarboxylate; [0275]DIEA for diisopropyl
ethylamine; [0276]DME for ethylene glycol dimethyl ether; [0277]DMF for
N,N-dimethyl formamide; [0278]ESI for electrospray ionization; [0279]Et
for ethyl; [0280]EtOAc for ethyl acetate; [0281]g for gram(s); [0282]h
for hour(s); [0283]HATU for
O-(7-Azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluoro-phosphate; [0284]HPLC for high-performance liquid
chromatography; [0285]Me for methyl; [0286]MeOH for methanol; [0287]mg
for milligram(s); [0288]min for minute(s); [0289]MS for mass
spectrometry; [0290]NMR for nuclear magnetic resonance; [0291]Ph for
phenyl; [0292]rt for room temperature; [0293]THF for tetrahydrofuran;
[0294]TLC for thin layer chromatography; [0295]PPh3 for
triphenylphosphine; [0296]tBOC or Boc for tert-butyloxy carbonyl;

Synthetic Methods

[0297]The compounds and processes of the present invention will be better
understood in connection with the following synthetic schemes that
illustrate the methods by which the compounds of the invention may be
prepared.

[0298]The targeted analogs were prepared from the common tripeptide
intermediates 1-6 and 1-8 and the like. Synthesis toward these versatile
intermediates began with the saponification of commercially available
Boc-hydroxyproline methyl ester (1-1) with lithium hydroxide in a 3:1:1
mixture of THF/MeOH/water to generate corresponding acid 1-2 (Scheme 1).
Subsequent coupling with the cyclopropyl-derived amino acid derivative
1-3 exploiting HATU afforded dipeptide 1-4. HCl-mediated Boc-deprotection
in dioxane yielded proline salt 1-5, which was further coupled with
Boc-tert-L-leucine to give the desired tripeptide 1-6. It is important to
note, that alternative amino acid derivatives can be used in either
coupling step to generate tripeptides analogous to 1-6, and therefore
ultimately produce multiple alternative pyridazinone analogs. Conversely,
1-5 could also be coupled to the olefin-containing amino acid 1-7
delivering a tripeptide that could be cyclized to intermediate 1-8
utilizing the first generation Hoveyda-Grubbs catalyst.

[0299]It is important to note, that although only the cis-hydroxyproline
series is shown in Scheme 1, the trans-hydroxyproline series can be
carried through an identical synthetic sequence. Both series are used in
order to generate the targeted HCV inhibitors.

[0300]Employing standard Mitsubobu protocols, cis-proline-containing
intermediates 1-6 and 1-8 can be transformed to the versatile
quinoxaline-containing compounds 2-1 and 2-2. Although this scheme is not
comprehensive, the chemistry portrayed therein serves as a general guide
toward multiple quinoxaline-derived species. For further details on the
Mitsunobu reaction, see O. Mitsunobu, Synthesis 1981, 1-28.

[0301]As stated above, both the cis-proline- and trans-proline-derived
intermediates were used in this study. Consequently, epi-1-6 and epi-1-8
can both be condensed with CDI, and the resulting product subjected to
various isoindolines to generate a variety of acyclic and cyclic
carbamates, represented by 3-1 and 3-2, respectively. As before, scheme 3
is not comprehensive, however the chemistry portrayed therein serves as a
general guide toward multiple carbamate-derived species.

[0302]Functionalization at the N-terminus can be carried out using a
two-step sequence (Scheme 4) beginning with HCl-mediated
Boc-deprotection. Once the amine salts (4-1 and 4-2) are generated, they
can then be condensed with an array of electrophiles under basic
conditions to produce alternative carbamates [--(C═O)--O--R1],
amides [--(C═O)--R1], ureas [--C(═O)--NH--R2], or
sulfonamides [--S(O)2--R1, --S(O)2NHR2], wherein
R1 and R2 are as previously defined.

[0303]The final steps to the targeted analogs of the present invention
include a saponification followed by the formation of the requisite
hydrazide moiety. This strategy was initiated with the treatment of the
ethyl esters 5-1 and 5-2 with lithium hydroxide in a 3:1:1 mixture of
THF/MeOH/water (Scheme 5a). Once the carboxylic acids 5-3 and 5-4 were
formed, they could transformed to the desired hydrazide compound in one
of two fashions: (I) the carboxylic acid is condensed with CDI, then
subjected to the fully functionalized hydrazide in the presence of DBU
(Scheme 5b), or (II) the carboxylic acid is condensed with CDI, subjected
to a 1M solution of hydrazine in THF, then the resulting free acyl
hydrazide is treated with various electrophiles (Scheme 5c). Examples of
this methodology are illustrated in, but not limited to, the conversion
of compounds represented by structure 5-1 and 5-2 to the
hydrazide-derived compounds represented by structures 5-5-5-8.

[0305]The compounds and processes of the present invention will be better
understood in connection with the following examples, which are intended
as illustrations only and not to limit the scope of the invention.
Various changes and modifications to the disclosed embodiments will be
apparent to those skilled in the art and such changes and modifications
including, without limitation, those relating to the chemical structures,
substituents, derivatives, formulations and/or methods of the invention
may be made without departing from the spirit of the invention and the
scope of the appended claims

[0306](Route 1) Synthesis of the Tri-Peptide Intermediates: (Note: this
Sequence was Also Carried Out Using the Trans-Hydroxy Proline Compound
Analogous to Structure 1a)

[0307]Step 1A. To a solution of commercially available
cis-L-hydroxyproline methyl ester (1a) (1.00 g, 4.1 mmol) in 165 ml of a
3:1:1 mixture of THF/MeOH/water at room temperature was added
LiOH.H2O (0.51 g, 12.2 mmol). The resulting heterogeneous reaction
was stirred at room temperature for 14 h, at which time the reaction was
concentrated to ˜1/5 of its original volume, then acidified with 6
M HCl(aq). This aqueous solution was then diluted with 20 mL brine and
extracted with DCM (4×50 mL). The organic washings were combined,
washed once with brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The resulting crude carboxylic acid 1b was carried
on without further purification.

[0308]Step 1B. Carboxylic acid 1b (4.08 mmol) was diluted with 50 mL of
DCM, cooled to 0° C., then consecutively treated with DIEA (4.1 g,
32.6 mmol), cyclopropyl-derived amino-acid hydrochloride salt 1c (0.78 g,
4.1 mmol), and HATU (1.9 g, 5.10 mmol). The reaction mixture was allowed
to warm to room temperature and closely monitored using mass
spectrometric analysis. Once the reaction was complete, it was
transferred to a 250 mL separatory funnel with 75 mL EtOAc, at which time
it was extracted with saturated aqueous NaHCO3 (2×20 ml) and
brine (2×20 ml). The organic phase was dried over anhydrous
Na2SO4, filtered, and then concentrated in vacuo. The residue
was purified by silica gel flash chromatography using gradient elution
with hexanes:EtOAc (5:1→3:1→1:1→1:2→1:5)
yielding the dipeptide 1d (0.826 g, 55%).

[0309]MS (ESI) m/z=369.3 (M+H).sup.+.

[0310]Step 1C. To neat dipeptide 1d was added 20 mL of a 4 M HCl solution
in dioxane. The resulting mixture was stirred at room temperature for 3
h. Once Boc-deprotection was complete, the excess HCl and organic solvent
was removed in vacuo. The resulting amino salt 1e was used without any
further purification.

[0311]MS (ESI) m/z=269.2 (M+H).sup.+.

[0312]Step 1D. Amine salt 1e (2.2 mmol) was diluted with 25 mL of DCM,
cooled to 0° C., then consecutively treated with DIEA (1.41 g,
11.2 mmol), Boc-tert-L-leucine (0.52 g, 2.2 mmol), and HATU (1.06 g, 2.8
mmol). The reaction mixture was allowed to warm to room temperature and
closely monitored using mass spectrometric analysis. Once the reaction
was complete, it was transferred to a 250 mL separatory funnel with 100
mL EtOAc, at which time it was extracted with saturated aqueous
NaHCO3 (2×20 ml) and brine (2×20 ml). The organic phase
was dried over anhydrous Na2SO4, filtered, and then
concentrated in vacuo. The residue was purified by silica gel flash
chromatography using gradient elution with hexanes:EtOAc
(5:1→3:1→1:1→1:2→1:5) yielding the desired
tripeptide intermediate 1f (113 g, 93%) as a white solid.

[0313]MS (ESI) m/z=482.4 (M+H).sup.+.

[0314]Step 1E. Amine salt 1e (2.24 mmol) was diluted with 25 mL of DCM,
cooled to 0° C., then consecutively treated with DIEA (1.41 g,
11.2 mmol), amino acid 1g (0.61 g, 2.24 mmol), and HATU (1.06 g, 2.80
mmol). The reaction mixture was allowed to warm to room temperature and
closely monitored using mass spectrometric analysis. Once the reaction
was complete, it was transferred to a 250 mL separatory funnel with 100
mL EtOAc, at which time it was extracted with saturated aqueous
NaHCO3 (2×20 ml) and brine (2×20 ml). The organic phase
was dried over anhydrous Na2SO4, filtered, and then
concentrated in vacuo. The residue was purified by silica gel flash
chromatography using gradient elution with hexanes:EtOAc
(5:1→3:1→1:1→1:2→1:5) yielding the desired
tripeptide intermediate 1h (1.0 g, 97%) as a white solid.

[0315]MS (ESI) m/z=544.84 (M+Na).sup.+.

[0316]Step 1F. A solution of the linear tripeptide 1f (1.5 g, 2.89 mmol)
in 200 ml dry DCM was deoxygenated by N2 bubbling (ca. 35 min).
Hoveyda's 1st generation catalyst (5 mol % eq.) was then added as a
solid. The reaction was refluxed under N2 atmosphere for 12 h. The
solvent was evaporated and the residue was purified by silica gel flash
chromatography using gradient elution with hexanes:EtOAc
(9:1→5:1→3:1→1:1→1:2→1:5). The cyclic
peptide precursor 1i was isolated as a white powder (1.2 g, 87%). For
further details of the synthetic methods employed to produce the cyclic
peptide precursor 1i, see WO 00/059929 (2000).

[0317]MS (ESI) m/z=516.28 (M+Na).sup.+.

Example 1

[0318]Compound of Formula IX, wherein

Step 2A.

[0320]To a cooled mixture of macrocyclic precursor 1i,
3-(thiophen-2-yl)-1H-quinoxalin-2-one (1.1 equiv.), and
triphenylphosphine (2 equiv.) in THF was added DIAD (2 equiv.) dropwise
at 0° C. The resulting mixture was held at 0° C. for 15 min
before being warmed to room temperature. After 1 h, the mixture was
concentrated under vacuum and the residue was purified by flash
chromatography eluting with 60% EtOAc in hexanes to give 2a as a clear
oil (100 mg, 99%).

[0324]Compound 2a (82 mg, 0.11 mmol) was treated with HCl (4 M in dioxane,
3 mL, 12 mmol). The reaction mixture was stirred at room temperature for
2 h until MS showed complete consumption of starting material. The
solvent was removed in vacuo and carried directly onto the next step
without further purification.

Step 2C.

[0326]The chloroformate reagent 2c was prepared by subjecting 0.22 mmol
cyclopentanol in THF (5 ml) to 0.45 mmol of phosgene in toluene (20%).
The resulting reaction mixture was stirred at room temperature for 2 h
and the solvent was then removed in vacuo. To the residue was added DCM
and the resulting mixture was subsequently concentrated in vacuo (repeat
2×) yielding chloroformate reagent 2c.

[0327]Amine salt 2b (ca. 0.11 mmol) was dissolved in DCM (3 mL) then
treated with cyclopentyl chloroformate (2c, 0.22 mmol) and DIEA (0.35 mL,
2 mmol). The reaction mixture was stirred for 2.5 h. Ethyl acetate (15
mL) was added to the solution, and the resulting reaction mixture was
consecutively washed with saturated aqueous NaHCO3 solution, water,
and brine. The organic layer was dried over anhydrous Na2SO4.
The organic phase was then filtered, concentrated in vacuo and purified
by flash chromatography (EtOAc/hexanes 1:2) to give 60.0 mg of the
cyclopentyl carbamate 2d.

[0328]MS (ESI) m/z 716.31 (M+H).sup.+.

Step 2D.

[0330]Carbamate 2d (60 mg, from above) was dissolved in THF/MeOH/H2O
(2:1:0.5) and subsequently subjected to lithium hydroxide (10 equiv.) at
room temperature for 20 h. The excess solvents were evaporated in vacuo,
and the resulting residue was diluted with water and acidified to pH 5.
The mixture was extracted with EtOAc (2×). The combined organic
extracts were washed once with brine, dried (MgSO4), filtered and
concentrated in vacuo to give carboxylic acid 2e, (42.0 mg, 55% for three
steps).

[0334]In a one-dram vial, carboxylic acid 2e (0.015 g, 0.022 mmol) was
dissolved in 0.75 mL DCM then treated with CDI (5.3 mg, 0.033 mmol). The
resulting mixture was then moved to a 45° C. oil bath and stirred
for 1 h. After cooling to rt, the vial was opened and nicotinic hydrazide
(8.6 mg, 0.066 mmol) was added along with DBU (9.2 μL, 0.066 mmol).
The vial was then purged N2, capped, and moved back to the
45° C. oil bath, where it was stirred for 3 h. After cooling, the
reaction mixture was loaded directly onto a plug of SiO2 and
purified via flash chromatography using EtOAc in hexanes
(20%→50%→95%) then 5% MeOH in EtOAc to yield the title
compound, (10.0 mg, 57%) as a white solid.

[0335]MS (ESI) m/z=807.3 (M+H).sup.+.

Example 2

[0336]Compound of Formula IX, wherein

[0337]Step 2E from above was followed using acetic hydrazide instead of
nicotinic hydrazide.

[0338]MS (ESI) m/z=744.3 (M+H).sup.+.

Example 3

[0339]Compound of Formula IX, wherein

[0340]Step 2E from above was followed using hydrazine (1.0 M in THF)
instead of nicotinic hydrazide (note: DBU was not needed for this
particular transformation).

[0341]MS (ESI) m/z=690.2 (M+H).sup.+.

Example 4

[0342]Compound of Formula IX, wherein

Step 3A.

[0344]Synthesis of the title compound was initiated by the condensation of
alcohol 3a (200 mg, 0.40 mmol--generated using trans-hydroxyproline and
the methodology outlined in Example 1) with CDI (79 mg, 0.49 mmol) in 5
mL DCM at rt. Once this coupling was complete as confirmed by MS
analysis, the isoindoline (145 mg, 1.21 mmol) was added and the resulting
mixture was stirred overnight. The reaction mixture was diluted with DCM
(20 mL) and washed with 1N aq. HCl (20 mL) and brine (20 mL). The organic
portion was then dried (Na2SO4), filtered, and concentrated in
vacuo. The crude oil was purified via flash chromatography (silica gel)
using dichloromethane/EtOAc/acetone (60:20:1) as eluent to afford the
corresponding carbamate (220 mg, 85%) as a white solid.

[0345]Once the carbamate portion was installed, ester hydrolysis was
carried out in standard fashion using LiOH in a THF/MeOH/water (1.5, 0.5,
0.5 mL, respectfully) solvent mixture. Upon completion, the reaction
mixture was diluted with 50 mL DCM and 5 mL water, which was acidified
with 1N aq. HCl. The layers were separated and the aqueous portion washed
three additional times with 10 mL DCM. The organic portions were combined
and washed once with brine (20 mL). Finally, the organic layer was dried
(Na2SO4), filtered, and concentrated in vacuo. The crude acid
3b was carried on to the coupling step without any further purification.

[0346]MS (ESI) m/z=611.3 (M+H).sup.+.

Step 3B.

[0348]Using 15 mg of carboxylic acid 3b, Step 2E from above was followed
using hydrazine (1.0 M in THF) instead of nicotinic hydrazide (note: DBU
was not needed for this particular transformation) to give the desired
compound.

[0392]The compounds of the present invention exhibit potent inhibitory
properties against the HCV NS3 protease. The following examples describe
assays in which the compounds of the present invention can be tested for
anti-HCV effects.

NS3/NS4a Protease Enzyme Assay

[0393]HCV protease activity and inhibition is assayed using an internally
quenched fluorogenic substrate. A DABCYL and an EDANS group are attached
to opposite ends of a short peptide. Quenching of the EDANS fluorescence
by the DABCYL group is relieved upon proteolytic cleavage. Fluorescence
is measured with a Molecular Devices Fluoromax (or equivalent) using an
excitation wavelength of 355 nm and an emission wavelength of 485 nm.

[0398]Cell lines, including Huh-11-7 or Huh 9-13, harboring HCV replicons
(Lohmann, et al Science 285:110-113, 1999) are seeded at 5×103
cells/well in 96 well plates and fed media containing DMEM (high
glucose), 10% fetal calf serum, penicillin-streptomycin and non-essential
amino acids. Cells are incubated in a 7.5% CO2 incubator at
37° C. At the end of the incubation period, total RNA is extracted
and purified from cells using Qiagen Rneasy 96 Kit (Catalog No. 74182).
To amplify the HCV RNA so that sufficient material can be detected by an
HCV specific probe (below), primers specific for HCV (below) mediate both
the reverse transcription of the HCV RNA and the amplification of the
cDNA by polymerase chain reaction (PCR) using the TaqMan One-Step RT-PCR
Master Mix Kit (Applied Biosystems catalog no. 4309169). The nucleotide
sequences of the RT-PCR primers, which are located in the NS5B region of
the HCV genome, are the following:

[0399]Detection of the RT-PCR product is accomplished using the Applied
Biosystems (ABI) Prism 7500 Sequence Detection System (SDS) that detects
the fluorescence that is emitted when the probe, which is labeled with a
fluorescence reporter dye and a quencher dye, is processed during the PCR
reaction. The increase in the amount of fluorescence is measured during
each cycle of PCR and reflects the increasing amount of RT-PCR product.
Specifically, quantification is based on the threshold cycle, where the
amplification plot crosses a defined fluorescence threshold. Comparison
of the threshold cycles of the sample with a known standard provides a
highly sensitive measure of relative template concentration in different
samples (ABI User Bulletin #2 Dec. 11, 1997). The data is analyzed using
the ABI SDS program version 1.7. The relative template concentration can
be converted to RNA copy numbers by employing a standard curve of HCV RNA
standards with known copy number (ABI User Bulletin #2 Dec. 11, 1997).

[0400]The RT-PCR product was detected using the following labeled probe:

[0401]The RT reaction is performed at 48° C. for 30 minutes
followed by PCR. Thermal cycler parameters used for the PCR reaction on
the ABI Prism 7500 Sequence Detection System are: one cycle at 95°
C., 10 minutes followed by 40 cycles each of which include one incubation
at 95° C. for 15 seconds and a second incubation for 60° C.
for 1 minute.

[0402]To normalize the data to an internal control molecule within the
cellular RNA, RT-PCR is performed on the cellular messenger RNA
glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The GAPDH copy number
is very stable in the cell lines used. GAPDH RT-PCR is performed on the
same exact RNA sample from which the HCV copy number is determined. The
GAPDH primers and probes, as well as the standards with which to
determine copy number, are contained in the ABI Pre-Developed TaqMan
Assay Kit (catalog no. 4310884E). The ratio of HCV/GAPDH RNA is used to
calculate the activity of compounds evaluated for inhibition of HCV RNA
replication.

[0404]The effect of a specific anti-viral compound on HCV replicon RNA
levels in Huh-11-7 or 9-13 cells is determined by comparing the amount of
HCV RNA normalized to GAPDH (e.g. the ratio of HCV/GAPDH) in the cells
exposed to compound versus cells exposed to the 0% inhibition and the
100% inhibition controls. Specifically, cells are seeded at
5×103 cells/well in a 96 well plate and are incubated either
with: 1) media containing 1% DMSO (0% inhibition control), 2) 100
international units, IU/ml Interferon-alpha 2b in media/1% DMSO or 3)
media/1% DMSO containing a fixed concentration of compound. 96 well
plates as described above are then incubated at 37° C. for 3 days
(primary screening assay) or 4 days (IC50 determination). Percent
inhibition is defined as:

[0409]The dose-response curve of the inhibitor is generated by adding
compound in serial, three-fold dilutions over three logs to wells
starting with the highest concentration of a specific compound at 10 uM
and ending with the lowest concentration of 0.0 uM. Further dilution
series (1 uM to 0.001 uM for example) is performed if the IC50 value is
not in the linear range of the curve. IC50 is determined based on the
IDBS Activity Base program using Microsoft Excel "XL Fit" in which A=100%
inhibition value (100 IU/ml Interferon-alpha 2b), B=0% inhibition control
value (media/1% DMSO) and C=midpoint of the curve as defined as
C=(B-A/2)+A. A, B and C values are expressed as the ratio of HCV
RNA/GAPDH RNA as determined for each sample in each well of a 96 well
plate as described above. For each plate the average of 4-6 wells are
used to define the 100% and 0% inhibition values.

[0410]In the above assays, representative compounds are found to have
activity.

[0411]Although the invention has been described with respect to various
preferred embodiments, it is not intended to be limited thereto, but
rather those skilled in the art will recognize that variations and
modifications may be made therein which are within the spirit of the
invention and the scope of the appended claims.